Evaluation of the optimal provision of formalin-fixed, paraffin-embedded material for reverse transcription-PCR in soft-tissue tumour diagnosis.

Abstract

Molecular genetic analysis is now a routine ancillary diagnostic modality to the histopathological diagnosis of soft-tissue neoplasms, many of which harbour characteristic gene fusions detectable by reverse transcription-PCR (RT-PCR). As the final diagnosis often depends on the molecular result, it is important to obtain the optimal yield of patient RNA.We assessed the most reliable method of providing formalin-fixed, paraffin-embedded material for optimal RNA yield by comparing three consecutive periods in which different preparations (5×10 μm scrolls, 5×5 μm sections and 1×10 μm sections) were used for RNA extraction for RT-PCR, with its technical success rate.For '2011', '2012' and '2013', RT-PCR technical failure rates were 13.4%, 4.4% and 7.9%, respectively. The percentage of failed referral cases was 71.4%, 85.7% and 31.3%, and the proportion of core biopsy to excision specimens was 3:15, 2:5 and 13:3.This study shows that the effectiveness of RNA extraction and purification is dependent on both specimen type and the tissue sectioning strategy. The failure rate has improved over recent years, particularly for large specimens as large numbers of thick 10 μm scrolls can saturate RNA extraction columns. In contrast, recent technical fails are more frequent in core biopsies, where 1×10 μm sections are insufficient for adequate RNA extraction. While previous technical fails occurred mostly in referred cases, this appears no longer the case due to the better fixation and processing of specimens in external surgical pathology departments because of the widespread recognition of the importance of molecular diagnostics as an important part of the patient pathway.